The molecular mechanisms of agonist-mediated endocytosis and intracellular membrane trafficking of G protein-coupled receptors (GPCRs) are not fully understood. Once internalized, the receptor either is recycled or degraded with each fate having different consequences. The recycled receptors will re-sensitize the response whereas degradation will diminish it. Identifying the mechanisms involved is important to our understanding of cellular responses to external stimuli. Several amino acid sequences in the beta2-adrenergic receptor (beta2-AR) have been shown to act as recognition signals for sorting proteins. It also has been shown that phosphorylation and ubiquitination have a role in the sorting of beta2-AR as well as several other GPCRs. Previously, we had shown that in contrast to the beta2-AR, the beta1-AR is not ubiquitinated. Using chimeric beta-ARs in which C-tails have been exchanged, we found that a beta1-AR with a beta2-AR C-tail is ubiquitinated but a beta2-AR with a beta1-AR C-tail is not. The relative roles of ubiquitination and phosphorylation in receptor sorting have not been established. A beta2-AR that is phosphorylated and internalized but not ubiquitinated may provide some insight into the relationship between phosphorylation and ubiquitination. As ubiquitination of beta2-AR is dependent on its C-tail, we replaced the three C-tail lysines with arginines (K348/372/375R) to generate the mutant 3K/R. Both wild type (WT) and mutant receptors have an N-terminal hemagglutin (HA) epitope tag and each was stably expressed in HEK 293 cells. Agonist-mediated ubiquitination of WT increased with time to a maximum at 4 h whereas ubiquitination of 3K/R was only 16% of WT. We also developed a cell-free assay using biotinylated ubiquitin and cell extracts. The receptors are immunoprecipitated with anti-C-tail or -HA antibodies and detected by HRP-streptavidin. The assay required ATP and extracts of agonist-stimulated cells as the receptors need to be phosphorylated in order to be ubiquitinated. Agonist increased the ubiquitination of WT whereas no ubiquitinated 3K/R was detected with or without agonist. We noticed that only a small fraction of the WT receptors was ubiquitinated at any time. We found similar results in other studies on ubiquitination of beta2-AR and different GPCRs. In cells continuously exposed to agonist, down-regulation of binding activity was observed after a 4-h delay. The losses were 40% for WT and 7% for 3K/R after 24 h. Similar losses were obtained when the receptors were detected by Western blotting with anti-C-tail antibodies.[unreadable] Using confocal microscopy, we observed that the agonist-stimulated endocytosis and intracellular trafficking of 3K/R was similar to that of beta1-AR as both appeared in small peripheral vesicles that became more punctate with time and did not accumulate in lysosomes. WT beta2-AR appeared in larger, perinuclear vesicles that coalesced into large aggregates and eventually fused with lysosomes. When the agonist was washed out, WT and 3K/R reappeared on the plasma membrane. Using a cell-surface binding assay, we quantified the internalization and recycling of WT and 3K/R and found the rates to be similar: t1/2 for WT, 2.65 and 9.64 min; and for 3K/R, 2.23 and 8.91 min. To further establish that the mutations in 3K/R did not affect its function, we compared its properties to those of WT beta2-AR. The Kd values for agonist and antagonist binding were not significantly different nor were the EC50 and Vmax values for stimulating adenylyl cyclase. Both were similarly desensitized in agonist-treated cells. Agonist-stimulated phosphorylation of both beta-ARs in intact cells was initially similar but after 2 min, 3K/R began to undergo dephosphorylation while WT phosphorylation continued to increase. Upon agonist removal, 3K/R was dephosphorylated 4 times faster than WT. As the increased rate was observed in cell-free and soluble assays, it appears to be intrinsic to the mutation and not a consequence of its distinct intracellular trafficking. Okadaic acid inhibited the dephosphorylation of both receptors in intact cells and in cell-free assays at concentrations that inhibit protein phosphatases PP2A and PP1. We confirmed that both are present in the cells by Western blotting. Using immunoprecipitated receptors from stimulated cells, we also showed that purified PP2A and PP1 dephosphorylated 3K/R more rapidly than WT beta2-AR. In okadaic acid-treated cells, lysosomal trafficking and degradation of 3K/R were increased almost to the levels of WT. Thus the increased rate of dephosphorylation of 3K/R is functionally relevant.[unreadable] Our data are consistent with a model of receptor trafficking that involves both phosphorylation and ubiquitination of the beta2-AR C-tail. Following phosphorylation and arrestin binding, the receptors are recruited to CCPs. Arrestins also attract ubiquitin ligases that ubiquitinate both arrestins and receptors. Ubiquitination of arrestins is transient and they are rapidly deubiquitinated by deubiquitinating enzymes and dissociate from the receptors which are endocytosed. Phosphorylated WT receptors that are not ubiquitinated enter an endocytic pathway where they are dephosphorylated and recycled. Receptors with both post-translational modifications are sorted to lysosomes where they are degraded. The ubiquitinated lysines and the phosphoserines function as a recognition signal for sorting proteins that direct the receptors to lysosomes. The ubiquitins also may protect the phosphoserines from protein phosphatases. As the rate of receptor internalization is 160-fold greater than that of down-regulation, only a small fraction of the internalized receptors are degraded and need to be ubiquitinated. As noted above, only a small fraction of WT beta2-AR is ubiquitinated at any time and is most likely due to the presence of deubiquitinating enzymes. As 3K/R lacks this signal and is rapidly dephosphorylated, it will not be targeted to lysosomes but instead recycled. Sorting of WT and 3K/R may occur early. Distinct populations of CCPs exist and GPCRs including beta2-AR enter only one of these populations and are excluded from the others. Phosphorylated beta2-AR (mainly WT) and dephosphorylated beta2-AR (mostly 3K/R) may enter two different populations of CCPs. Upon endocytosis, the two populations of CCPs follow different endocytic routes. Further dephosphorylation and/or sorting can occur in several intracellular compartments such as early, sorting, recycling and late endosomes. This will allow phosphorylated receptors to be dephosphorylated and recycled except for the ubiquitinated WT receptors which will be sorted to lysosomes. Phosphorylation and ubiquitination appear to be involved in the intracellular sorting of beta2-AR between recycling and degradation. Phosphorylation may to be a sufficient signal for degradation but only when dephosphorylation is prevented. Ubiquitination insures that the signal is maintained.